Abstract This project focuses on the design and evaluation of prodrugs with dual pharmacological selectivity, combining molecularly targeted mode of action with a tissue-specific, active uptake process (uptake-1) to enhance drug delivery to aggressive neuroendrocrine neoplasms, including high-risk neuroblastoma (NB) ? the deadliest extracranial pediatric solid tumor currently lacking effective treatment options. Norepinephrine transporter (NET) driving accumulation of norepinephrine and its functional analogs is expressed by most solid tumors developing from sympathoadrenal precursor cells. However, tumor radiotherapy targeted to NET has shown limited efficiency, while causing serious adverse effects due to significant off-target distribution and extensive damage to healthy tissues. Centered on a dual-selective experimental drug delivery strategy integrating the uptake-1 process with a replication-dependent mode of drug action to confine the pharmacological effect to proliferative tumor cells expressing NET, this project aims to evaluate and optimize a pharmacotherapeutic approach designed to effectively combat refractory disease not responding to existing treatments, while minimizing toxicity to healthy organs and tissues. In our proof-of-concept experiments, a tripartite prodrug design integrating NET affinity with unique molecular targeting of a potent and selective topoisomerase I inhibitor was shown to be essential for achieving sustained intratumoral drug presence and markedly extended survival in clinically relevant models of aggressive neuroblastoma. Guided by these results, we hypothesize that dual-selective pharmacotherapy using NET-targeted prodrugs can provide a selective, safe and efficient way of treating different forms of high-risk disease. We also hypothesize that potency and selectivity of this approach will be enhanced by combining it with clinically proven small-molecule agents modulating tissue-specific expression of NET. These hypotheses will be tested by pursuing the following specific aims: Aim 1 studies will focus on comparative evaluation of polymeric carrier-linked prodrug constructs with regard to their cell uptake and growth inhibitory effects on primary NB cells and cell lines with different phenotypes, as a function of their molecular design and the potentiating action of the NET expression enhancing agents; Aim 2 and Aim 3 studies will comparatively evaluate the biodistribution profiles and therapeutic effectiveness of a series of tripartite prodrugs, with the goal to identify and optimize key construction variables, to establish feasibility of pharmacologically modulating NET expression for improving drug delivery and treatment outcomes, and to examine the roles of tumor phenotype and disease status in clinically relevant models of aggressive NB. The proposed research focusing on NET-targeted prodrugs equipped with dual pharmacological selectivity is significant by informing the development of a new strategy for treating aggressive NB and other refractory cancers.